The central theme of the Research Project is the study of the mechanisms involved in the determination of the prion strain and phenotype in human prion diseases. The rationale of the proposal is based on the widely accepted notion that there is a close correlation between the conformation of the scrapie prion protein (PrPSc or prion strain) and the disease phenotype. Therefore,the mechanisms determining PrPSc conformation and disease phenotype can be traced to the same origin. Five specific aims are proposed. Specific Aim 1 investigates the role played by glycans in phenotypic determination. Disease phenotypes and PrPSo characteristics will be investigated in PrP glycosylation incompetent transgenic (Tg) mice expressing human PrP (humanized mice) following inoculation with PrPSc from a subtype of sporadic Creutzfeldt-Jakobdisease (sCJD) characterized by phenotypic and PrPSc features likely to be related to the presence of unusual glycans. Specific Aim 2 addresses the issue of the infectivity and competenceto reproduce the phenotype of human PrPSc in relation to its state of aggregation. PrPSc oligomers as well as small and large aggregates will be separated by size exclusion chromatography and will be individually inoculated to humanized mice. Infectivity and disease phenotype will be determined. Specific Aims 3 and 4 take advantage of the novel technique of protein misfolding cyclic amplification (PMCA) to assess: i) the role of the PrP genotype in prion strain determination and ii) whether PMCA can be used to circumvent species barriers. The characteristics of PMCA-generated PrPSc species will be compared after using normal or cellular PrP (PrPc) differing in amino acid sequence at position 129 as substrate for the replication. PrPSc species that are difficult to transmit by bioassay, such as the PrPSc associated with chronic wasting disease and bovine spongiform encephalopathy, will be replicated by PMCA using humanized Tg mice as donors of the PrPc needed for the conversion and inoculated to humanized mice. Transmissibility of the novel bovine amyloidotic spongiform encephalopathy (BASE) to humanized mice will also be attempted. Specific Aim 5 is dedicated to the characterization of a novel human prion disease we recently identified that, remarkably, is associated with protease-sensitive PrPSc and lack mutations in the PrP gene coding region despite an often positive family history. The proposed research will advance the current understanding of mechanisms of prion strain and phenotypic determination. Furthermore, it may develop procedures that overcomethe limitation of the bioassay and lead to the expression of new phenotypes relevant to prion zoonoses, opening newavenues for research on prion diseases.